Structural Biology of the Apical Bile Acid Transporter

描述（由申请人提供）：顶钠依赖性胆汁酸转运蛋白（ASBT）在胆盐的肠肝循环、胆固醇稳态中起关键作用，并作为高胆固醇药物的分子靶点。虽然转运体序列是已知的，但对其膜拓扑结构存在争议，对ASBT的结构功能和配体结合域知之甚少。拟开展ASBT的结构生物学研究。利用结合分子生物学和计算生物学的新方法，我们的长期目标是描绘ASBT的三维结构、配体结合域和细胞运输机制。以下具体目标将被解决：(1)使用一系列拓扑扫描方法定义ASBT的膜拓扑；(2)构建ASBT的综合结构和预测模型，将结构点突变与配体亲和力和转运变化联系起来；(3)通过定点诱变确定ASBT的功能区域；我们开发了一种计算机辅助的定点诱变方法来探测ASBT蛋白中与配体和钠相互作用有关的氨基酸残基；(4)质谱法确定ASBT的配体结合域；我们将采用选择性光亲和标记来确定配体结合肽序列。这些研究获得的信息将大大增加我们对驱动胆汁酸运输的结构相互作用的理解，并进一步了解溶质载体蛋白的结构。此外，它可能有助于未来开发针对高胆固醇血症和相关心血管疾病的特定治疗策略。

DESCRIPTION (provided by applicant): The apical sodium-dependent bile acid transporter (ASBT) plays a key role in the enterohepatic recycling of bile salts, cholesterol homeostasis, and serves as a molecular target for hypercholesterolemic agents. Although the transporter sequence is known, there is controversy about its membrane topology and very little is known about ASBT structure-function and ligand binding domains. The proposed research will focus on the structural biology of ASBT. Using a novel approach that combines molecular and computational biology our long-term goal is to delineate the three-dimensional structure, ligand-binding domains, and cellular transport mechanism of ASBT. The following specific aims will be addressed: (1) define the membrane topology of ASBT using a series of topology scanning approaches; (2) Construct a comprehensive structural and predictive model of ASBT that can correlate structural point mutations to changes in ligand affinity and transport; (3) Define the functional regions of ASBT by site-directed mutagenesis; we have developed a computer-assisted site-directed mutagenesis approach to probe ASBT protein for amino acid residues implicated in ligand and sodium interactions; (4) Determine the ligand binding domains of ASBT by mass spectrometry; we will employ selective photoaffinity labels to determine ligand-binding peptide sequences. Information gained by these studies will significantly increase our understanding of the structural interactions that drive bile acid transport and further our structural knowledge of solute carrier proteins in general. Additionally, it may aid future development of specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.